Protein degradation by the proteasome must be tightly regulated, as it controls events ranging from cell cycle progression to cell death. The 26S proteasome is composed of a 20S catalytic core particle (CP) that is capped at either end by a 19S regulatory particle (RP). Whereas the CP contains the enzymatic activity responsible for proteolyzing protein substrates, the commitment to protein degradation is determined by RP components, which recognize and process substrates prior to their passage into the CP. For most proteasome substrates, ubiquitination is prerequisite to their degradation, as their initial interaction with the proteasome is through RP ubiquitin receptors, S5a and Rpn13. Substrates are subsequently deubiquitinated by three deubiquitinating enzymes and unfolded by a heterohexomeric ring of ATPases. These activities prepare substrates for entry through a narrow chamber leading to the catalytic center of the proteasome's CP. Since not all ubiquitinated proteins that dock into the proteasome are ultimately degraded, there is a great deal of interest in understanding the mechanistic details of substrate processing in the RP. Our long-term goal is to define how substrates are processed by the 19S regulatory particle of the proteasome. We focus this proposal on the first stage of this process, namely substrate recognition and deubiquitination, through our studies of Rpn13 and S5a. We study how Rpn13 is docked into the proteasome and pursue preliminary data that indicates it to be activated by this localization. We have devised a novel strategy to study Rpn13 and S5a in the context of the RP and test a working model of their coordinated binding to ubiquitinated substrates. To achieve our research goals, we use functional assays, including those that test the ubiquitin binding capacity of purified proteasome species and ubiquitin chain deconjugation by Uch37, as well as a variety of biophysical methods, especially NMR spectroscopy. Ultimately, the completion of the proposed research will provide fundamental information on distinct roles assumed by Rpn13 and S5a that extend beyond simply docking ubiquitinated proteins into the proteasome. PUBLIC HEALTH RELEVANCE: This proposal focuses on the proteasome, a biomachine that removes proteins at the appropriate time for proper cellular function. The proteasome is a major therapeutic target against neurodegenerative diseases and cancer and its general inhibition by Velcade is used to treat multiple myeloma. The outcome of this proposal will afford fundamental information on how the proteasome degrades its substrates and factors involved in determining whether ubiquitinated proteins are fated for degradation.